6-O sulfated polysaccharides and methods of preparation thereof

ABSTRACT

Disclosed are methods of 6-O-sulfating glucosaminyl N-acetylglucosamine residues (GlcNAc) in a polysaccharide preparation and methods of converting anticoagulant-inactive heparan sulfate to anticoagulant-active heparan sulfate and substantially pure polysaccharide preparations may by such methods. Also disclosed is a mutant CHO cell which hyper-produces anticoagulant-active heparan sulfate. Methods for elucidating the sequence of activity of enzymes in a biosynthetic pathway are provided.

This application is a National Stage Application of PCT InternationalApplication PCT/US02/10172, filed Mar. 28, 2002, which claims priorityfrom U.S. provisional Application No. 60/279,523, filed Mar. 28, 2001,and U.S. provisional Application No. 60/316,289, filed Aug. 30, 2001,which are incorporated herein by reference.

RELATED APPLICATIONS

Benefit of priority is claimed to U.S. Provisional Application SerialNo. 60/279,523, which was filed on Mar. 28, 2001, and U.S. ProvisionalApplication Serial No. 60/316,289, which was filed on Aug. 30, 2001, thedisclosures of which are herein incorporated by reference.

GOVERNMENT SUPPORT

Work described herein was supported by National Institutes of HealthGrants 5-P01-HL41484, 5-R01-HL58479, and GM-50573. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the biosynthesis ofglucosaminoglycans, and in particular to 6-O-sulfating polysaccharides.

BACKGROUND

Heparin/heparan sulfate (HS) is a linear polymer covalently attached tothe protein cores of proteoglycans, which are abundant and ubiquitouslyexpressed in almost all animal cells. HS is assembled by the action of alarge family of enzymes that catalyze the following series of reactions:chain polymerization comprising the alternating addition ofN-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) residues;GlcNAc N-deacetylation and N-sulfation; glucuronic acid epimerization toL-iduronic acid (IdoUA); 2-O-sulfation of uronic acid residues; and 3-O-and 6-O-sulfation of glucosaminyl residues.

The interaction between HS and various proteins occur in highly sulfatedregions of the HS. Furthermore, the specificity of any HS-proteininteraction is largely dictated by arrangement of sulfates along the HSchain. For example, the pentasaccharide sequence,GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S, represents the minimumsequence required for antithrombin (AT) binding, where the 3S(3-O-sulfate) and 6S (6-O-sulfate) groups constitute the most criticalelements involved in the binding (12-16). The AT-HS complex has potentanticoagulant properties. Several enzymes involved in anticoagulantheparan sulfate (HS^(act)) biosynthesis have been purified and cloned.For example, glucosaminyl 3-O sulfotransferase (3-OST) and glucosaminyl6-O-sulfotransferase (6-OST) proteins have been purified and cloned(17,18). Multiple isoforms of 6-OST and 3-OST proteins have beenisolated and shown to have tissue-specific expression patterns anddistinct substrate specificities.

Two different sulfated domains are present in HS, namely, the NS domainand NAc/NS domain (40,41). The NS domains consist of contiguousiduronosyl N-sulfoglucosamine units, while the NAc/NS domain consists ofalternating N-acetylated and N-sulfated disaccharides. Acceptorspecifcities of 6-OST-1, 6-OST-2, and 6-OST-3 using N-sulfated heparosanand desulfated re-N-sulfated heparin as substrate, indicated that thesulfation of position 6 of the N-sulfoglucosamine residues in the NSdomain is catalyzed by 6-OST-1, 2A, 2B, and 3 and the sulfation ofposition 6 of the N-sulfoglucosamine residues in the NA/NS domain arecatalyzed by 6-OST-2 and 6-OST-3 (2).

Tissue-specific and developmentally regulated expression of the HSbiosynthetic enzymes and enzyme isoforms produce HS chains with specificsequences (1-3). This microheterogeneity enables HS to interact with abroad array of protein ligands that modulate a wide range of biologicalfunctions in development, differentiation, homeostasis, andbacterial/viral entry (reviewed in refs (4-11)). Syntheticpolysaccharides which possess such specific sequences may be used tomodulate such biological functions.

Heparin preparations, particularly preparations comprising HS^(act),have been used clinically as anticoagulant therapeutics for theprevention and treatment of thrombotic disease. HS^(act) preparationshave also been used to maintain blood fluidity in extracoporeal orcorporeal medical devices such as dialysis devices and stents,respectively.

SUMMARY OF THE INVENTION

In one aspect, the present invention features methods of transferring asulfate on to the 6-O position of a GlcNAc sugar residue in apolysaccharide preparation, the method comprising the steps of (a)providing a polysaccharide preparation having GlcNAc sugar residues, and(b) contacting the polysaccharide preparation with 6-OST protein in thepresence of a sulfate donor under conditions which permit the 6-OSTprotein to add a sulfate to the 6-O-position of a GlcNAc sugar residue.In preferred embodiments the sulfate donor is PAPS.

In some embodiments, the polysaccharide preparation comprises glucuronicacid (GlcUA) residues; GlcUA-GlcNAc sugar residues; disaccharidesselected from the consisting of GlcUA/IdoUA-GlcNS, IdoUA2S-GlcNS, andGlcUA-GlcNS3S. In some preferred embodiments, the polysaccharidepreparation includes the pentasaccharide sequence of the antithrombinbinding motif, namely, GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S.

In some embodiments, the polysaccharide preparation includes precursorsaccharides for the antithrombin binding motif for example.GlcNAc/NS-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S, GlcNAc/NS6S-GlcUA-GlcNS3S±-IdoUA2S-GlcNS6S, GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS.Particularly preferred precursors include IdoA/GlcA-GlcNAc6S,IdoA/GlcA-GlcNS6S, and IdoA2S-GlcNS6S.

In some embodiments the 6-OST protein is a recombinant protein producedin an expression system such as baculovirus cells, bacteria cells,mammalian cells, or yeast cells. In some preferred embodiments the 6-OSTis human 6-OST, however, 6-OST from other mammals may also be used. Inparticularly preferred embodiments, the 6-OST protein comprises apolypeptide selected from the group consisting of (a) human 6-OST-1 (SEQID NO. 3); (b) human 6-OST-2A (SEQ ID NO. 4); (c) human 6-OST-2B (SEQ IDNO. 5); (d) human 6-OST-3 (SEQ ID NO. 6); (e) an allelic or speciesvariant of any of a-d; and (f) a functional fragment of any one of a-d.

In some embodiments, the sulfation reaction mixture comprising at leastone chloride salt, and the pH is between 6.5 and 7.5 In preferredembodiments, the 6-OST is contacted with the polysaccharide preparationprotein in the presence of a sulfate donor for at least 20 minutes. Inother embodiments, the reaction proceeds overnight.

In another aspect, the present invention features method of enrichingthe portion of HS^(act) present in a polysaccharide preparationcomprising: (a) providing a 3-O-sulfated polysaccharide preparation; and(b) contacting the preparation with 6-OST protein in the presence of asulfate donor under conditions, which permit the 6-OST protein to add asulfate the 6-O-position of a GlcNAc sugar residue, wherein, step (b)occurs concurrent with or subsequent to step (a). In preferredembodiments, the sulfate donor is PAPS. In some embodiments, thepolysaccharide preparation is derived from heparan; however, thepolysaccharides may be derived from other sources of polysaccharidesknown in the art.

In some embodiments, the 3-O-sulfated polysaccharide preparation isderived from a cell that expresses 3-OST-1, in alternative embodiments,the 3-O-sulfated polysaccharide preparation is prepared by contactingHS^(inact) with 3-OST-1 protein (SEQ ID NO 2), allelic or speciesvariant, or functional fragments of 3-OST-1.

In preferred embodiments, the percentage of HS^(act) present in thepolysaccharide preparation following step (b) is greater than 50%. Inparticularly preferred embodiments, the percentage of HS^(act) presentin the polysaccharide preparation following step (b) is greater than70%.

Preferred polysaccharide preparations for use in the methods of theinvention comprise N-acetylglucosamine (GlcNAc) and glucuronic acid(GlcUA) residues. Particularly preferred polysaccharide preparations foruse in the methods of the invention comprise GlcUA/IdoUA-GlcNS,GlcUA-GlcNAc, IdoUA2S-GlcNS, and GlcUA-GlcNS3S.

In another aspect, the present invention features, a mutant CHO cell(“hyper-producer”) that produces between 28% and 50% HS^(act). Inpreferred embodiment, the hyper-producer produces 50% HS^(act) relativeto total HS produced by the cell. The mutant CHO cell may be produced bya method comprising: (a) transforming a CHO cell with multiple copies of3-OST-1, allelic or species variant or functional fragment thereof; (b)mutagenizing the cell obtained in step (a); (c) isolating a mutant cellfrom step (b) which fails to product HS^(act); and (d) transforming thecell obtained in step (c) with 6-OST. In particularly preferredembodiments, the 6-OST protein comprises a polypeptide selected from thegroup consisting of (a) human 6-OST-1 (SEQ ID NO. 3); (b) human 6-OST-2A(SEQ ID NO. 4); (c) human 6-OST-2B (SEQ ID NO. 5); (d) human 6-OST-3(SEQ ID NO. 6); (e) an allelic or species variant of any of a-d; and (f)a functional fragment of any one of a-d.

In another aspect, the present invention features, a method ofelucidating the sequence of components in a biosynthetic pathwaycomprising the steps of: (a) providing a target cell which expresses atleast the upstream components of the biosynthetic pathway; (b)transforming the target cell with multiple copies of an isolatedbiosynthetic pathway downstream gene; (c) mutagenizing the transformedtarget cell; and (d) identifying transformed and mutagenized targetcells that fail to express the phenotype characteristic of thebiosynthetic pathway. In some embodiments, that method further comprisesthe step of (e) correcting the step (d) cells. In such embodiments, thecorrecting step may comprise inserting an upstream gene into the cellsof step (d). The upstream gene may be a cDNA, genomic DNA, or afunctional fragment thereof. In preferred embodiments, the cells of step(d) are transformed with a pool of preselected cDNAs for components ofthe biosynthetic pathway, for example, a cDNA library derived from acell that expresses the characteristic non-mutant phenotype.

In some embodiments, the correcting step may comprise contacting thecells of step (d) with the gene product of an upstream gene. Inalternative embodiments, the correcting step may comprise contacting thecells of step (d) with the mRNA, cDNA, genomic DNA, or a functionalfragment thereof for the upstream gene.

In some embodiments, the method further comprises the step of isolatingthe cells from step (d), analyzing the cells of step (d), and/orisolating the upstream gene in the biosynthetic pathway.

In some embodiments, the mutagenesis step comprises a mutagenesistechnique selected from the group consisting of chemical mutagenesis ionradiation, and ultraviolet radiation. The step of identifying the genecDNA may comprise complementation analysis, Northern blot analysis,Southern blot analysis, and/or Western blot analysis. In preferredembodiments, upstream gene may be isolated using PCR or any othertechnique known in the art.

In another aspect, the present invention features methods of reducingthrombin activity in a medical device comprising the step of coating themedical device with any of the substantially pure preparations and/orpreparations enriched for HS^(act) disclosed herein. In preferredembodiments the medical device is an extracorporeal or intracorporealdevice that contacts blood.

These and other objects, along with advantages and features of theinvention disclosed herein, will be made more apparent from thedescription, drawings, and claims that follow.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts IPRP-HPLC of [³⁵S]sulfate metabolic labeled HSdisaccharides. The IPRP-HPLC was performed as follows. [³⁵S]sulfatemetabolically labeled HS from parental wild-type, mutant, and correctantwere isolated and digested with a mixture of heparitinases. Theresulting disaccharides were separated on a Bio-Gel P2 column and werethen further resolved by IPRP-HPLC with appropriate internalstandards. 1. ΔUA-GlcNS; 2. ΔUA-GlcNAc6S; 3. ΔUA-GlcNS6S; 4.ΔUA2S-GlcNS; 5. ΔUA2S-GlcNS6S. Blue tracer, mutant; red tracer,correctant; black broken tracer, wild-type. The broken line indicatesthe gradient of acetonitrile.

FIG. 2 depicts IPRP-HPLC of 6-OST-1 and [³⁵S]PAPS-labeled HSdisaccharides. The IPRP-HPLC was performed as follows. Cold HS from3-OST-1 expressing CHO wild-type and precursor mutant were in vitrolabeled with purified baculovirus expressed 6-OST-1 in the presence[³⁵S]PAPS for 20 min. (A) or overnight (B). HS[³⁵S] was isolated anddigested with a mixture of heparitinases. The resulting disaccharideswere separated on a Bio-Gel P2 column and further resolved by IPRP-HPLCwith internal standards. 1. ΔUA-GlcNAc6S; 2. ΔUA-GlcNS6S; 3.ΔUA2S-GlcNS6S. Solid tracer, mutant; broken tracer, wild-type. Thebroken line indicates the gradient of acetonitrile.

FIG. 3 depicts Bio-Gel P6 fractionation of digested HS. The Bio-Gel P6fractionation was performed as follows. 6-O-[³⁵S]sulfate tagged [³H]HSfrom mutant were digested with 1 mU heparitinase I for 1 hour. HS^(act)oligosaccharides were obtained by AT-affinity chromatography. HS^(act)oligosaccharides were then treated with low pH nitrous acid and thenNABH₄ reduced, or treated with heparitinase I, II, and heparinase wereanalyzed by Bio-Gel P6 chromatography. The fractions indicated werepooled for further analysis. A, 6-O-[³⁵S]sulfate tagged mutant HS^(act)oligosaccharides; B, 6-O-[³⁵S]sulfate tagged mutant HS^(act)oligosaccharides treated with low pH nitrous acid and NaBH₄; C,6-O-[³⁵S]sulfate tagged mutant HS^(act) oligosaccharides digested withheparitinases. n=the number of monosaccharide units in each peak.

FIG. 4 depicts IPRP-HPLC of 6-O-sulfate tagged HS^(act) di- andtetrasaccharides. The IPRP-HPLC was performed as follows. In vitro6-O-sulfated and AT-affinity purified [³H]HS^(act) oligosaccharides weredigested with a mixture of heparitinases. The resulting di- andtetrasaccharides were separated on a Bio-Gel P6 column (FIG. 3C). (A),tetrasaccharides collected from FIG. 3C, peak 1:ΔUA-GlcNAc6³⁵S-GlcUA-GlcNS3S, peak 2: ΔUA-GlcNAc6³⁵S-GlcUA-GlcNS3S6³⁵S;(B), disaccharides of the digested tetrasaccharides in the presence ofHIP peptide; peak 1: ΔUA-GlcNAc6³⁵S, peak 2: ΔUA-GlcNS3S6³⁵S; (C),disaccharides collected from FIG. 3C, peak 1: ΔUA-GlcNS6³⁵S, peak 2:ΔUA2S-GlcNS6³⁵S. The broken line indicates the gradient of acetonitrile.

FIG. 5 depicts dual-color fluorescence flow cytometric analysis of AT(A, C, E, and G) and FGF-2 (B. D, F, and H) binding to wild-type,mutant, and 6-OST-1 correctant. CHO wild-type (A and B); wild-type CHOcell clone with 3 copies of 3-OST-1, (C and D), mutant cell clone with 3copies of 3-OST-1 (E and F), and 6-OST-1 correctant of the mutant (G andH) were double-labeled with fluorescein-AT (A, C, E, and G) and Alexa594-FGF-2 (B, D, F, and H) and subjected to dual-color FACS.

FIG. 6 depicts HPLC anion-exchange chromatography of GAGs. The HPLCanion-exchange was performed as follows. [³H]GlcN-Labeled GAG chainsfrom wild-type and mutant were isolated by protease digestion andβ-elimination. Samples were analyzed by HPLC anion-exchangechromatography. Solid tracer, mutant; broken tracer, wild-type. Thebroken line indicates the concentration gradient of sodium chloride.

FIG. 7 illustrates one embodiment of a method for elucidating theHS^(act) biosynthetic pathway. In this embodiment, using recombinantretroviral transduction, the human heparan sulfate (HS)3-O-sulfotransferase 1 (3-OST-1) gene was transduced into Chinesehamster ovary (CHO) cells. 3-OST-1 expression gives rise to CHO cellswith the ability to produce anticoagulant HS (HS^(act)). A cell linecontaining 3 copies of 3-OST-1 was chosen by Southern analysis. Afterchemical mutagenesis of this cell line, FGF-2 binding positive and ATbinding negative mutant cells were FACS sorted and cloned. The advantageof having 3 copies of 3-OST-1 is that upstream genes that areresponsible for generating specific HS precursor structures can besought after chemical mutagenesis without being concerned with the lossof 3-OST-1. FGF-2 selection is employed to make certain that the mutantcells still make HS.

FIG. 8 depicts ΔUA-GlcNS3 S disaccharide structure as determination bycapillary IPRP-HPLC coupled with mass spectrometry. The IPRP-HPLC-MSanalysis was performed as follows. Cold HS chain from wild-type CHOcells were labeled with 3-OST-1 plus PAP³⁴S. Purified HS was digestedwith a combination of 1 mU of each heparitinase I, heparitinase II,heparitinase IV, and heparinase in the presence of 0.5 mg/mlheparin/heparan sulfate interacting protein (HIP) peptide. 0.5 μg ofdigested HS was injected into capillary IPRP-HPLC coupled with MS. PanelA, UV tracer of capillary IPRP-HPLC from 35.85 to 39.71 min., peak Bcontains both ΔUA-GlcNS6S and ΔUA-GlcNS3 S, and peak D containsΔUA2S-GlcNS; panel B, negative polarity MS spectra from 37.44 to 38.17min.; which equals UV peak from 36.64 to 37.37 min.; panel C,amplification of m/z 494.0 to 501.0 region from panel B; panel D,negative polarity MS spectra from 38.17 to 39.06 min.; which equals UVpeak from 37.37 to 38.26 min.; panel E, amplification of m/z 494.0 to501.0 region from panel D.

DETAILED DESCRIPTION

Before proceeding further with a detailed description of the currentlypreferred embodiments of the instant invention, an explanation ofcertain terms and phrases will be provided. Accordingly, it isunderstood that each of the terms set forth is defined herein at leastas follows:

Anticoagulant heparan sulfate (HS^(act)). As used herein the term“anticoagulant heparan sulfate” or the abbreviation “HS^(act)” means asulfated HS comprising the pentasaccharide binding site forantithrombin, namely, GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S.HS^(act) may be purified from a pool of polysaccharides by any meansknown in the art, for example, AT-affinity chromatography. Theanticoagulant activity of a sample may be quantitated using thetechniques disclosed herein, or alternatively using an assay known inthe art, for example, the Coatest Heparin assay manufactured byChromogenix, Milan, Italy.

Anticoagulant-inactive heparan sulfate (HS^(inact)) As used herein theterm “anticoagulant-inactive heparan sulfate” or the abbreviation“HS^(inact)” means a sulfated HS lacking the pentasaccharide bindingsite for antithrombin, namely,GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S. Anticoagulant-inactiveheparan sulfate may also be identified and quantitated using thetechniques disclosed herein or any assay known in the art, for example,the Coatest Heparin assay manufactured by Chromogenix, Milan, Italy.

Enriched. As used herein with regard to particular polysaccharidestructures within a polysaccharide preparation, the term “enriched”means that the proportion of the polysaccharide structure in apolysaccharide preparation is statistically significantly greater thanthe proportion of the polysaccharide structure in naturally-occurring,untreated polysaccharide preparation. The polysaccharide preparations ofthe invention are enriched for 6-OST-1-sulfated polysaccharides orHS^(act) approximately 10-100 fold. For example, whereas the percentageof 6-OST-sulfated polysaccharide in a typical, unenriched preparation isbetween 0%-3%, the percentage of 6-OST-sulfated polysaccharide in theenriched polysaccharide preparations of the invention is betweenapproximately 5-9%. Likewise, whereas the percentage of HS^(act) in atypical, produced by cells culture in vitro is between approximately0-1%, the percentage of HS^(act) in the enriched polysaccharidepreparations of the invention derived from the hyper-producing mutantCHO cell of the invention is between approximately 28-50%.

Heparan sulfate. As used herein, the term “heparan sulfate” or theabbreviation “HS” means a polysaccharide made up of repeateddisaccharide units D-glucuronic acid or L-iduronic acid linked toN-acetyl or N-sulfated D-glucosamine. The polysaccharide is modified toa variable extent by sulfation of the 2-O-position of GlcA and IdoAresidues, and the 6-O- and 3-O-positions of GlcN residues andacetylation or de-acetylation of the nitrogen of GlcN residues.Therefore, this definition encompasses all of the glycosaminoglycancompounds variously referred to as heparan(s), heparan sulfate(s),heparin(s), heparin sulfate(s), heparitin(s), heparitin sulfate(s),heparanoid(s), heparosan(s). The heparan molecules may be pureglycosaminoglycans or can be linked to other molecules, including otherpolymers such as proteins, and lipids, or small molecules.

3-O-Sulfotransferases. As used herein, the term “3-O-Sulfotransferases”refers to the family of proteins that are responsible for the additionof sulfate groups at the 3-OH position of glucosamine in HS. Theseenzymes are present as several isoforms expressed from different genesat different levels in various tissues and cells. The 3-OSTs act tomodify HS late in its biosynthesis (reviewed by Lindahl et al., 1998)and each isoform recognizes as substrate glucosamine residues in regionsof the HS chain that have specific, but different, prior modifications,including epimerization and sulfation at other nearby positions (Liu etal., 1999). Thus, different 3-OSTs generate different potentialprotein-binding sites in HS.

3-OST-1. As used herein, the term “3-O-sulfotransferase-1” or theabbreviation “3-OST-1” refers to the particular isoform of the6-O-Sulfotransferase family designated as “1”. 3-OST-1 is described indetail in WO 99/22005, which is herein incorporated by reference in itsentirety. As used herein 3-OST-1 may refer to the nucleic acidcomprising the 3-OST-1 gene (SEQ. ID NO. 1) or the protein (SEQ. ID NO.2). Whether the term is applied to nucleic acids or polypeptide, it isintended to embrace minimal sequences encoding functional fragments of3-OST-1. In general, a functional fragment comprises the minimumsegments required for transfer of a sulfate to the 3-O position of HS.Accordingly, a functional fragment may omit, for example, leadersequences that are present in full-length 3-OST-1. WO 99/22005 providesfurther guidance regarding which segments of full-length 3-OST-1 nucleicacids and polypeptides comprise functional fragments.

6-O-Sulfotransferases. As used herein, the term “6-O-Sulfotransferases”refers to members of the family of 6-OSTs are responsible for theaddition of sulfate groups at the 6-OH position of glucosamine in HS.These enzymes are present as several isoforms expressed from differentgenes at different levels in various tissues and cells. As is the casewith the 3-OSTs, the 6-OSTs act to modify HS late in its biosynthesisand each isoform recognizes as substrate glucosamine residues in regionsof the HS chain that have specific, but different, prior modifications,including epimerization and sulfation at other nearby positions (Liu etal., 1999).

As used herein 6-OST may refer to nucleic acids or polypeptidescomprising human 6-OST-1, -2A, -2B, and -3. Whether the term I appliedto nucleic acids or polypeptide, it is intended to embrace allelic andspecies variants, as well as minimal segment(s) required for transfer ofa sulfate to the 6-O position of an HS preparation and, in particular,GlcNAc residues of HS. Accordingly, a functional fragment may omit, forexample, the transmembrane and/or leader sequences that are present inthe full-length protein.

Substantially pure. As used herein with respect to polysaccharidepreparations, the term “substantially pure” means a preparation whichcontains at least 60% (by dab weight) the polysaccharide of interest,exclusive of the weight of other intentionally included compounds.Preferably the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by dry weight the polysaccharideof interest, exclusive of the weight of other intentionally includedcompounds. Purity can be measured by any appropriate method, e.g.,column chromatography, gel electrophoresis, amino acid compositionalanalysis or HPLC analysis. If a preparation intentionally includes twoor more different polysaccharides of the invention, a “substantiallypure” preparation means a preparation in which the total dry weight ofthe polysaccharide of the invention is at least 60% of the total dryweight, exclusive of the weight of other intentionally includedcompounds. Preferably, for such preparations containing two or morepolysaccharides of the invention, the total weight of thepolysaccharides of the invention should be at least 75%, more preferablyat least 90%, and most preferably at least 99%, of the total dry weightof the preparation, exclusive of the weight of other intentionallyincluded compounds. Thus, if the polysaccharides of the invention aremixed with one or more other compounds (e.g., diluents, detergents,excipients, salts, sugars, lipids) for purposes of administration,stability, storage, and the like, the weight of such other compounds isignored in the calculation of the purity of the preparation.Furthermore, when the polysaccharide is a proteoglycan, the proteincomponent of the proteoglycan is excluded for purposes of calculatingpurity.

Transformation. As used herein, transformation means any method ofintroducing exogenous a nucleic acid into a cell including, but notlimited to, transformation, transfection, electroporation,microinjection, direct injection of naked nucleic acid,particle-mediated delivery, viral-mediated transduction or any othermeans of delivering a nucleic acid into a host cell which results intransient or stable expression of the nucleic acid or integration of thenucleic acid into the genome of the host cell or descendant thereof.

Variant. As used herein, “variant” DNA molecules are DNA moleculescontaining minor changes in a native 6-OST sequence, i.e., changes inwhich one or more nucleotides of a native 6-OST sequence is deleted,added, and/or substituted, preferably while substantially maintaining a6-OST biological activity. Variant DNA molecules can be produced, forexample, by standard DNA mutagenesis techniques or by chemicallysynthesizing the variant DNA molecule or a portion thereof. Suchvariants preferably do not change the reading frame of theprotein-coding region of the nucleic acid and preferably encode aprotein having no change, only a minor reduction, or an increase in6-OST biological function. Amino-acid substitutions are preferablysubstitutions of single amino-acid residues. DNA insertions arepreferably of about 1 to 10 contiguous nucleotides and deletions arepreferably of about 1 to 30 contiguous nucleotides. Insertions anddeletions are preferably insertions or deletions from an end of theprotein-coding or non-coding sequence and are preferably made inadjacent base pairs. Substitutions, deletions, insertions or anycombination thereof can be combined to arrive at a final construct.Preferably, variant nucleic acids according to the present invention are“silent” or “conservative” variants. “Silent” variants are variants of anative 6-OST sequence or a homolog thereof in which there has been asubstitution of one or more base pairs but no change in the amino-acidsequence of the polypeptide encoded by the sequence. “Conservative”variants are variants of the native 6-OST sequence or a homolog thereofin which at least one codon in the protein-coding region of the gene hasbeen changed, resulting in a conservative change in one or more aminoacid residues of the polypeptide encoded by the nucleic-acid sequence,i.e., an amino acid substitution. In all instances, variants of thenaturally-occurring 6-OST, as described above, must be tested forbiological activity as described below. Specifically, they must have theability to add a sulfate to the 6-OH position of a sugar residue in HS.

The present invention depends, in part on the discovery that (i) 6-OSTis limiting enzyme in the HS^(act) biosynthetic pathway when 3-OST-1 isnon-limiting; (ii) 6-OST can add 6-O-sulfate to GlcNAc residues,including the critical 6-O-sulfate in the antithrombin binding motif ofHS; and (iii) both 3-O- or 6-O-sulfation may be the final step inHS^(act) biosynthesis. Thus, the present invention provides methods ofsynthesizing oligosaccharides comprising GlcNAc6S, preparations enrichedfor HS^(act), and methods of making such preparations using 6-OST.

Methods of 6-O-Sulfating Polysaccharides

In one aspect, the present invention provides methods for 6-O-sulfatingsaccharide residues within a preparation of polysaccharides in which thepolysaccharides includes a GlcNAc sugar residue. These methods comprisecontacting a polysaccharide preparation with 6-OST protein in thepresence of a sulfate donor under conditions which permit the 6-OST toconvert the GlcNAc sugar residue to GlcNAc6S. In particularly preferredembodiments, the 6-OST protein comprises a polypeptide selected from thegroup consisting of (a) human 6-OST-1 (SEQ ID NO. 3); (b) human 6-OST-2A(SEQ ID NO. 4); (c) human 6-OST-2B (SEQ ID NO. 5); (d) human 6-OST-3(SEQ ID NO. 6); (e) an allelic or species variant of any of a-d; and (f)a functional fragment of any one of a-d. In preferred embodiments, thesulfate donor is 3′-phospho-adenosine 5′-phosphosulfate (PAPS).

In another aspect, the present invention provides methods of producingHS^(act) by contacting a 3-O-sulfated polysaccharide preparation with6-OST protein. These methods are based upon the discovery that6-O-sulfation can occur after 3-O-sulfation in HS^(act) biosynthesis. Inparticular embodiments, a GlcNAc sugar residue which comprises a part ofan HS^(act) precursor sequence is 6-O-sulfated. In some embodiments, thetarget polysaccharide comprises part of an HS^(act) precursor sequence,for example, IdoA/GlcA-GlcNAc6S, IdoA/GlcA-GlcNS6S, and IdoA2S-GlcNS6S.In some preferred embodiments, the target polysaccharide is 3-O-sulfatedprior to or concurrently with 6-O-sulfation.

In another aspect, the present invention also provides for means ofenriching the AT-binding fraction of a heparan sulfate pool (i.e.,increasing the portion of HS^(act)) by contacting a polysaccharidepreparation with 6-OST protein in the presence of a sulfate donor underconditions which permit the 6-OST to convert HS^(inact) to HS^(act). Inpreferred embodiments, the sulfate donor is 3′-phospho-adenosine5′-phosphosulfate (PAPS). Conversion of the HS^(act) precursor pool toHS^(act) using the methods of the invention is particularly useful inthe production of anticoagulant heparan sulfate products which haveclinical applications as therapeutics, for example, as an agent to treator prevent thrombotic disease. Anticoagulant heparan sulfate productsmay alternatively be used as agents to maintain blood flow in medicaldevices, for example, dialysis machines. In general, the preparationsenriched for HS^(act) disclosed herein may be use in any application inwhich anticoagulant HS is employed.

In yet another aspect, the present invention provides a recombinant cellline that expresses enhanced levels of HS^(act). In vitro cell culturesproduce between 0%-1% HS^(act). However; the corrected mutant(“correctant or hyper-producer”) created by transforming CHO cellsmultiple copies of 3-OST-1, followed mutigenization of the resultanttransformant and transformation with 6-OST-1 has been shown to expressbetween 28%-50% HS^(act). This represents a significant improvement overthe percentage of HS^(act) produced by any cell line known to applicantsat the time of filing.

The 6-O-sulfated preparations and the HS^(act) produced by the methodsof the invention are useful as therapeutic agents to treat and/orprevent any condition improved by administration of an anticoagulant,for example, thrombotic disease. These compositions may also be used tocoat the surfaces of extracorporeal medical devices (e.g., dialysistubing) or intracorporeal devices (e.g., transplants, stents or otherprosthetic implants) to reduce blood clotting on those surfaces.

Practice of the invention will be still more fully understood from theexamples, which are presented herein for illustration only and shall notbe construed as limiting the invention in any way.

Example 1 6-O-sulfation of HS In Vitro

6-O-sulfation of glucosaminylglyans in vitro may be accomplished in anymanner known in the art. As a skilled artisan would recognize, a6-O-sulfation reaction requires a 6-OST protein, or functional fragmentthereof, a target polysaccharide, a sulfate donor (preferably PAPS), anda pH in the range of 6.5-7.5 (preferably a pH of about 7.0). Thus, in apreferred procedure, the reaction mixture contains 50 mM MES (pH 7.0),1% (w/v) Triton X-100, 5 mM MnCl₂, 5 mM MgCl₂, 2.5 mM CaCl₂, 0.075 mg/mlprotamine chloride, 1.5 mg/ml BSA, either metabolically labeled [³⁵S]HSor non-radioactive HS chains, cold PAPS (0.5 mM) or [³⁵S]PAPS (25 μM,2×10⁷ cpm), and 70 ng of purified baculovirus-expressed human 6-OST-1 ina final volume of 50 μl. The mixtures may be incubated either 20 minutesor overnight at 37° C., and 200 μg of chondroitin sulfate C added. HSchains are purified by phenol/chloroform extraction and anion exchangechromatography on 0.25-ml columns of DEAE-Sephacel packed in 1 mlsyringes (20). After ethanol precipitation, the pellets are washed with75% ethanol, dried briefly under vacuum, and dissolved in water forfurther analysis.

Example 2 6-OST Generate HS^(act) In Vitro

To explain the difference between 6-OST substrate specificity observedin vivo and previously reported specifcities, 6-OST-1 was expressed andpurified in bacteria and baculovirus. The purified proteins were used tosulfate HS³⁵S derived from the precursor mutant and wild-type CHO cells.Specifically, precursor mutant HS chains were treated with baculovirusexpressed, 3-OST-1 protein, 6-OST-1 protein, or both proteins in thepresence of cold PAPS. HS^(act) was isolated by AT-affinity purificationand the percentage of HS^(act) was quantitated. The results are shownbelow in Table 1. As Table 1 shows, the yield of HS^(act) resulting from6-OST-1 treatment of precursor mutant HS chain (51%) was similar to thatof the CHO wild-type (64%) even though 6-O-sulfation is severelydecreased in the precursor mutant (FIG. 1).

TABLE 1 Percentage of [S³⁵]HS^(act) 3-OST-1 and Control 3-OST-1 6-OST-16-OST-1 Wild-type CHO 26% 40% 64% 70% Precursor Mutant  7% 12% 51% 64%

Example 3 6-OST-1 Sulfation Generates Three Kinds of 6-O-ContainingDisaccharides In Vitro

To localize where 6-OST-1 adds 6S residues along the HS chains, equalamounts of HS from 3-OST-1 expressing wild-type and precursor mutantwere in vitro labeled with purified baculovirus expressed 6-OST-1 in thepresence of [³⁵S]PAPS either for 20 minutes or overnight. Only ˜⅓ asmuch radioactivity was incorporated into the HS derived from 3-OST-1expressing CHO cells as compared to the HS derived from the precursormutant cells. [S³⁵]HS was isolated and digested with a mixture ofheparitinases. The resulting disaccharides (accounting for ˜94% of [³⁵S]counts) were separated on a Bio-Gel P2 column and further resolved byIPRP-HPLC with appropriate internal standards (FIG. 2, mutant, solidtracer; wild-type, broken tracer). As FIG. 2 shows, 6-OST-1 not onlyadded a 6S group on GlcNS, but 6-OST-1 also 6S group on GlcNAc residuesin both the 3-OST-1 expressing CHO HS and precursor mutant HS.

As summarized below in Table 2, more ΔUA-GlcNAc6³⁵S and ΔUA-GlcNS6³⁵Sdisaccharides were observed from reactions run overnight than after just20 minutes. 6-O-sulfate incorporation was 10 times higher fromincubation with baculovirus expressed 6-OST-1 than bacteria expressed6-OST-1. However, overnight labeling using bacterial 6-OST-1 generatedthree 6-O-sulfated disaccharides in the following proportions:ΔUA-GlcNAc6³⁵S (25%), ΔUA-GlcNS6³⁵S (20%), and ΔUA2S-GlcNS6³⁵S (55%).This ratio of 6-O-sulfated disaccharides is comparable to the ratioobserved in baculovirus 6-OST-1 overnight labeled disaccharides (FIG. 2,panel B).

TABLE 2 Overnight 20 Minute Incubation Incubation ΔUA-GlcNAc6³⁵S 29% 18%ΔUA-GlcNS6³⁵S 18% 12% ΔUA2S-GlcNS6³⁵S 53% 70%

Example 4 Contribution of 6-OST in Generation of HS^(act)Oligosaccharides

To further locate the 6-O-sulfate addition in AT-binding HS^(act)oligosaccharides, cold mutant HS chains were treated with purifiedBaculovirus expressed 6-OST-1 with [³⁵S]PAPS overnight. Afterheparitinase I digestion, HS^(act) oligosaccharides were affinitypurified (7% of 6-O-[³⁵S]sulfate-labeled HS^(total)). The HS^(act)oligosaccharides were then treated with low pH nitrous acid that cleavesN-sulfated residues, and a combination of heparitinases that cleaves3-O-sulfate containing sugar into tetrasaccharides and all other sugarsinto disaccharides. Treated and untreated HS^(act) oligosaccharides wererun on Bio Gel P6 columns (FIG. 3). Di- and tetrasaccharides werecollected from enzyme and low pH nitrous treated samples as indicated.The tetrasaccharides resistant to a combination of heparitinases I, II,and heparinase digestion represented the 3-O-sulfate containingtetrasaccharides as reported earlier (20,33). The presence of similaramounts of tetrasaccharides from both nitrous and enzyme degradationsuggests the 3-O-containing tetrasaccharides have the structures,UA±2S-GlcNAc6³⁵ S-GlcUA-GlcNS3 S±6³⁵S. To prove this, thetetrasaccharides (FIG. 4A) collected from enzyme digestion (FIG. 3C)were further digested into disaccharides (FIG. 4B) with heparitinase Iin the presence of HIP peptide (the same method as shown in FIG. 8).IPRP-HPLC profiles of 6-O-sulfate tagged HS^(act) di- andtetrasaccharides from FIG. 3C were shown in FIG. 8. Table 3 summarizesthe 6-O-[³⁵S]sulfate-labeled disaccharide compositions calculated basedon the HPLC data (FIG. 4).

TABLE 3 Percentage of [S³⁵]HS^(act) 3-OST-1 and Control 3-OST-1 6-OST-16-OST-1 Wild-type CHO 26% 40% 64% 70% Precursor Mutant  7% 12% 51% 64%

In HS^(act) oligosaccharides, 6-OST adds 6-O-sulfates not only atGlcUA/IdoUA-GlcNS, GlcUA-GlcNAc, and IdoUA2S-GlcNS, but also atGlcUA-GlcNS3S. These results show that 6-OST is the enzyme that not onlyputs the critical 6-O-sulfate group in HS^(act) oligosaccharides, butalso other 6-O-sulfate groups in HS^(act) oligosaccharides as well.3-OST-1 and 6-OST are therefore the critical enzymes for the generationof HS^(act).

3-OST-1, usually existing in limited amounts, acts upon HS^(act)precursor to produce HS^(act) and upon HS^(inact) precursor to produce3-O-sulfated HS^(inact) (17,19). When 3-OST-1 is no longer limiting, thecapacity for HS^(act) generation is determined by the abundance ofHS^(act) precursors (20). Since in vitro 3-O-sulfation can transformHS^(inact) into HS^(act), it was previously believed that 3-O-sulfationis the final modification step during biosynthesis of HS^(act).Surprisingly, in vitro 6-O-sulfation was also shown to transform3-O-sulfate containing HS^(inact) into HS^(act). Thus, the presentdisclosure provides methods of enriching a polysaccharide preparation ofHS^(act) by contacting a HS^(inact) with 6-O-sulfate protein and asulfate donor under conditions which permit 6-OST-1 to sulfate a GlcNAcsugar residue.

Example 5 6-OST-1 Corrected Mutant Makes HS, 50% of which is HS^(act)

To determine whether the diminished 6-OST activity in the precursormutant caused the precursor mutant's deficiency in AT binding, precursormutant was transduced with 6-OST-1 cDNA. To create the 6-OST-1 cDNA, CHO6-OST-1 coding region was amplified and sequenced from the CHO-K1quick-clone cDNA library by PCR. Since only partial 6-OST-1 codingsequence from CHO cells has been reported (32), the complete CHO 6-OST-1sequence was deposited in Genbank (accession number: AB006180). Stable6-OST-1 transfectants were screened by FACS. Specifically, the cellswere labeled with fluorescein-AT and Alexa 594-FGF-2 and then subjectedto dual-color FACS. The FACS analysis for a correctant cell is shown inFIG. 5, at panels G and H. The correctants with high AT binding affinitywere single-cell-cloned. HS[³⁵S] from correctants was isolated byAT-affinity chromatography and analyzed The correctant produced HS andHS^(act). Surprisingly, approximately between 28% and 50% of total HSproduced by the correctant was HS^(act). The only cultured cell known tothe applicants at the time of the filing the instant applicationproduces approximately 0%-1%^(HSact.)

Example 6 Correctant Cells Produce a Greater Percentage of GlcNac6S andGlcNS6S Residues In Vivo than Either the Wild-Type CHO Cells Expressing3-OST-1 and the Precursor Mutant Cells

The disaccharide composition of the HS derived from correctant cells wasshown to a comprise a greater percentage of GlcNAc6S and GlcNS6Sresidues than CHO cells or mutant cells as follows. HS[³⁵S] from 3-OST-1expressing CHO cells, precursor mutant cells and correctant cells wasisolated and digested with a mixture of heparitinases. The resultingdisaccharides were independently separated on a Bio-Gel P2 column andfurther resolved by IPRP-HPLC (FIG. 6). The relative percentages are setforth below.

TABLE 4 Disaccharide Wild-type Precursor mutant Correctant GlcNAc6S 7%5%  9% GlcNS6S 9% 4% 13%Precursor Mutant

The present disclosure also provides a method, which constitutes ageneral approach for defining and obtaining components of biosyntheticpathways when (1) the gene for a downstream or terminal biosyntheticenzyme has been isolated, and (2) an assay for the downstream product(s)is available. This method of delineating biosynthetic pathways comprisesplacing multiple copies of the gene for a down-stream component of thepathway (i.e., a component acting at or near the end of a biosyntheticpathway) into a target cell line to produce a multi-expresser;mutagenizing the multi-expresser to obtain mutants deficient inup-stream component (i.e., components that generate precursor structuresearlier in the pathway than the downstream component); and analyzing theprecursor mutants. In some embodiments the method further comprises“correcting” the precursor mutant, for example, by transducing themutant with the gene encoding a putative precursor protein. Thetransduction may be accomplished using one or more previously identifiedcomponents of the biosynthetic pathway or using a “shotgun” libraryapproach. In other embodiments, the correction may entail contacting theprecursor mutant with the gene products of the biosynthetic pathway andscreening for the phenotype of the wild-type, for example, ligandbinding.

The advantage of a cell line containing multiple copies of a terminal ordownstream gene product is that the activity of the downstream geneproduct remains intact following mutagenesis, therefore, upstream geneproducts will determine the phenotype of the mutagenized cell. Thus, thepresent invention provides methods of generating mutants specificallydefective for upstream gene products, as well as, methods for isolatingdownstream components and delineating biosynthetic pathways.

Example 7 Creation of Precursor Mutant

To elucidate HS^(act) biosynthesis, mutants defective in the formationof HS^(act) precursors were created. Chinese hamster ovary (CHO) cellswere selected as the target cell because wild-type CHO cells produceHS^(inact) but not HS^(act) (presumably, due to lack of HS3-O-Sulfotransferase-1 (3-OST-1) expression). Furthermore, a series ofHS biosynthetic mutants have been successfully made in CHO cells(23-28).

The 3-OST-1 gene, which was presumed to be the terminal enzyme in theHS^(act) biosynthetic pathway, was introduced into CHO cells byretroviral transduction (29). 3-OST-1 expression gave rise to CHO cellswith the ability to produce HS^(act). A CHO cell line containing 3copies of 3-OST-1 (referred to herein as “3-OST-1 expressing CHO cells,“3-OST-1 triple mutant” or “multi-expresser”) was selected for furtheranalysis and experimentation. The 3-OST-1 triple mutant was subjected tochemical mutagenesis. Cells positive for the desired knockout phenotype,specifically, positive for HS expression (selected by FGF-2 binding) andnegative for HS^(act) expression (selected by AT binding), wereidentified and isolated (FIG. 5, panels E and F). This cell line isreferred to herein as the “6-OST-1 deficient mutant” or “precursormutant.”

The precursor mutant disclosed herein, which makes decreased amounts of6-O-sulfated residues, is defective in AT binding (FIG. SE) due todecreased 6-O-sulfotransferase activities. The defect in this mutant hasbeen corrected, both in vivo (by transduction with a 6-OST-1 gene) andin vitro (by contacting HS with 6-OST-1 protein).

Example 8 Correction of the Precursor Mutant

The 6-OST-1 sulfate defect of the 6-OST-1 deficient mutant was corrected(i.e., the phenotype of the parental cell line was recovered) bytransduction with 6-O-sulfotransferase-1 gene (FIG. 5, panels G and H).The resultant cell line (the “Correctant”) produced HS, 50% of which isHS^(act). Previously reported cell lines have been observed to produceless than 1% ^(HSact.) This represents the highest percentage ofHS^(act) production by any reported cell line. Thus, the presentinvention provides for a cell line that produces high yields ofHS^(act), as well as methods of efficiently producing HS^(act). Thiscell line (termed “hyper-producer”) expresses approximately 28%-50% ofHS^(act) relative to HS^(total.)

Example 9 GAGs from Precursor Defective Mutant and Wild-Type CHO Cellshave Similar Charge Densities

GAGs from the 3-OST-1 expressing CHO cells and precursor mutant cellswere isolated and analyzed by biosynthetic labeling studies using[6-³H]GlcN. HPLC anion-exchange analysis of the [³H]GAG chains from theprecursor mutant resolved HS (0.31-0.50 M NaCl) from chondroitin sulfate(0.52-0.60 M NaCl) (FIG. 6). The GAG chains from the 3-OST-1 expressingCHO cells (FIG. 6, solid tracer) resolved into a similar profile to thatof the precursor mutant (FIG. 6, broken tracer). This result impliesthat the HS from the precursor mutant and the 3-OST-1 expressing CHOcells have similar charge densities charge density and therefore, thedecrease in AT-binding activity observed in the precursor mutant may beattributed to structural changes in the HS, possibly due to differencesin degree of sulfation.

Example 10 Precursor Mutant Makes Less 6-O-Sulfate ContainingDisaccharides than 3-OST-1 Expressing CHO Cells

Since HS from the precursor mutant has similar charge density to that ofthe 3-OST-1 expressing CHO cells, the decrease in AT binding in theprecursor mutant was expected to correlate with a change in thestructure of the HS chains. The GAGs synthesized by the 3-OST-1expressing CHO cells and precursor mutant were analyzed by biosyntheticlabeling studies using [³⁵S]sulfate. The 3-OST-1 expressing CHO cellsand precursor mutant cells produced the same amount of [³⁵S]HS and bothsamples contained ˜70% HS and ˜30% chondroitin sulfate (data not shown).This ratio of HS to chondroitin sulfate is consistent with the resultshown in FIG. 6 (wherein HS accounted for 68% of the GAGs in theprecursor mutant, and HS accounted for 66% of the GAGs in the 3-OST-1expressing CHO cells). [³⁵S]sulfate labeled HS chains from the 3-OST-1expressing CHO cells and precursor mutant cells were then digested witha mixture of heparitinases. The resulting disaccharides (representingapproximately 93% of total [³⁵S]sulfate counts) were separated on aBio-Gel P2 column and further resolved by IPRP-HPLC with appropriateinternal standards. As Table 5 shows, the precursor mutant cellsproduced reduced amounts of 6-O-sulfated disaccharides relative to the3-OST-1 expressing CHO cells.

TABLE 5 Disaccharide Composition 3-OST-1 expressing Disaccharide CHOcells Precursor Mutant Correctant ΔUA-GlcNS 30% 35% 27% ΔUA-GlcNAc6S  7% 5%  9% ΔUA-GlcNS6S  9%  4% 13% ΔUA2S-GlcNS 20% 36% 22% ΔUA2S-GlcNS6S34% 19% 29%

Example 11 Precursor Mutant and 3-OST-Expressing CHO Cells Express6-OST-1 mRNA, but not 6-OST-2 mRNA or 6-OST-3 mRNA

In order to explain the reduced levels of 6-O-sulfate containingdisaccharides in the precursor mutant, 6-OST-1 isoform expression in3-OST-1 expressing CHO cells was compared with 6-OST-1 isoformexpression in the precursor mutant cells. Human 6-OST-1, 6-OST-2, and6-OST-3 cDNA were used as probes in Northern blot and RT-PCR studies.Northern analysis indicated that the precursor mutant and the 3-OST-1expressing CHO cells have the same level of 6-OST-1 mRNA. However, no6-OST-2 or 6-OST-3 mRNA was detected in either the 3-OST-1 expressingCHO cells or the precursor mutant cells, indicating that CHO cellsexpress 6-OST-1 only.

The expression pattern for 6-OST isoforms in CHO cells was confirmedusing RT-PCR analysis of 3-OST-1 expressing CHO cells and precursormutant cells. One set of PCR primers for 6-OST-1, three sets of PCRprimers for 6-OST-2, and two sets of PCR primers for 6-OST-3 were usedto evaluate mRNA expression of the 6-OST isoforms. The same level of6-OST RT-PCR products was observed for both 3-OST-1 expressing CHO cellsand the precursor mutant cells; however, no RT-PCR products wereobserved in either cell from the three sets of 6-OST-2 RT-PCR reactionsand two sets of 6-OST-3 RT-PCR reactions. The Northern blot and RT-PCRanalyses described above demonstrate that CHO cells express 6-OST-1, butnot 6-OST-2 or 6-OST-3.

Example 12 The Coding Region of 6-OST-1 in the Precursor Mutant ContainsNo Point Mutations

Northern blot and RT-PCR analysis indicated that the precursor mutantcells and the 3-OST-1 expressing CHO cells express similar levels of6-OST-1 mRNA, however, as described in greater detail below, the levelof 6-OST-1 activity in the precursor mutant is lower than the level ofactivity in the 3-OST-1 expressing CHO cells. This observation raisedthe possibility that the precursor mutant CHO cells might have one ormore point mutation(s) in 6-OST-1 gene that diminishes the level of6-OST sulfotransferase activity. The coding regions of 6-OST-1 RT-PCRproducts from the mutant were double-strand-sequenced and no pointmutation was observed compared to wild-type 6-OST-1. Thus, thediminished level of 6-OST activity observed is not due to a defect inthe 6-OST-1 gene in the precursor mutant.

Example 13 Precursor Mutant has Decreased 6-O-SulfotransferaseActivities Compared to Wild-Type CHO Cells

FACS analysis showed that the precursor mutant cells were defective inAT binding (FIG. 5, panel E). The coding sequence of 6-OST-1 was notmutated and 6-OST-1 mRNA expression levels were normal; however,disaccharide compositional studies demonstrated that the precursormutant made less 6-O-sulfated residues in vivo than wild-type CHO cells.

The 6-O-sulfotransferase activity of the precursor mutant was evaluatedin vitro. Crude cell homogenates from wild-type CHO cells and precursormutant CHO cells served as the source of 6-OST-1 enzyme. HS derived fromwild-type CHO cells, N,O-desulfated, re-N-sulfated heparin(CDSNS-heparin), and 6-O-desulfated heparin was incubated with 6-OST-1enzyme from wild-type CHO cells and precursor mutant in the presence ofa sulfate donor. The resulting reaction products were digested by acombination of heparitinases, followed by Bio Gel P2 chromatography. Thedisaccharides collected were then subjected to IPRP-HPLC analysis. Both2-O-[³⁵S]sulfate (control) and 6-O-[³⁵S]sulfate labeled disaccharidesresulting from the 6-OST-1 enzymes were quantitated.2-O-sulfotransferase activity was similar in precursor mutant cells(118±3 pmol/min/mg) and the wild-type CHO cells (122±2 pmol/min/mg) whenCDSNS-heparin was used as substrate (not shown). However, a 30% to 39%reduction of 6-O-sulfotransferase activity was observed in the precursormutant relative to the wild-type CHO cells with all three substrates(Table 6).

TABLE 6 6-O-sulfotransferase activity (pmol/min/mg) % reduction ofwild-type activity Wild-type Precursor in precursor Substrate CHO Mutantmutant HS (CHO K1) 5.6 ± 0.3 3.9 ± 0.4 30% 6-O-desulfated heparin 4.4 ±0.3 2.7 ± 0.5 39% CDSNS-heparin 11 ± 2  7 ± 1 38%

Example 14 Size Exclusion Chromatography Coupled with Mass Spectrometryis Effective for Compositional Analysis of Oligosaccharides

Mass spectrometric detectors produce far more information thanconventional UV or fluorescent detectors and allows the monosaccharidecomposition of individual components to be determined (39). Introducingstable isotope PAP³⁴S into the 3-O-position of HS by pure 3-OST-1, a3-O-sulfate containing disaccharide with a unique mass was identifiedusing a combination of capillary IPRP-HPLC coupled with massspectrometry. The method consumes 0.5 μg of total HS for separating anddetecting different HS disaccharides. This method provides a practicalway of accomplishing HS disaccharide analysis of general HS samples fromcells or tissues without radioisotope labeling. Furthermore,biologically inactive HS oligosaccharides could be treated in vitro withdifferent pure sulfotransferases plus stable sulfur isotope PAPS (e.g.,PAP³³S and PAP³⁴S). The different stable isotope tagged biologicallyactive oligosaccharides could then be sequenced by a combination ofcapillary IPRP-HPLC for separation and mass spectrometry. In thismanner, biologically critical regions can be pinpointed and sequenced.

Capillary IPRP-HPLC coupled with mass spectrometry. Heparin moleculesexhibiting a high affinity for a synthetic peptide (CRPKAKAKAKAKDQTK)(SEQ ID NO. 7) mimicking a heparin-binding domain of heparin interactingprotein (HIP) also show an extremely high affinity for AT (37). It wasexpected that inclusion of this small peptide in the heparitinasedigestion solution would protect 3-O-[³⁵S]sulfate labeled HS fromdegrading into tetrasaccharide. Theoretically, HIP peptide-protected, ATbinding HS oligosaccharides would be recovered. However, in the presenceof the HIP peptide, all the 3-O-[³⁵S]sulfate labeled sugars weredegraded into disaccharides instead of oligosaccharides ortetrasaccharides as judged by their elution position on Bio-Gel P2 andtheir unique elution positions on IPRP-HPLC (the major 3-O-[³⁵S]sulfatecontaining disaccharides eluted right before ΔUA-GlcNS6S disaccharidestandard). Because there is no ΔUA-GlcNS3S standard reported, thestructure was verified. Stable, isotope PAP³⁴S was made. The PAP³⁴S (99%isotope purity determined by ES-MS) was prepared by incubating ATP andstable isotope Na₂ ³⁴SO₄ (Isonics Corp.) with ATP sulfurylase (Sigma),adenosine 5′-phosphosulfate kinase (a generous gift from Dr. Irwin H.Segel), and inorganic pyrophosphatase (Sigma) (38). HS chains fromwild-type CHO cells were labeled with pure 3-OST-1 plus PAP³⁴S. Acapillary IPRP-HPLC (LC Packings) method for separating HS disaccharideswas developed. This method is similar to conventional IPRP-HPLC (29)except using 5 mM dibutylamine as an ion pairing reagent (Sigma), andthen coupled it to an ESI-TOF-MS (Mariner Workstation, PerSeptiveBiosystems, Inc.) to detect the mass of each disaccharide eluted. Six HSdisaccharide standards from Seikagaku were separated by capillary HPLCand detected by negative polarity ESI-MS. The accuracy of the ES-MS is±0.001 m/z unit after calibration with the molecular standard setssupplied by the manufacture (Bis TBA, Heptadecaflurononanoic acid,Perflurotetradecanoic acid). 3-O-³⁴S-labeled HS was digested with acombination of 1 mU of each heparitinase I, heparitinase II,heparitinase IV, and heparinase in the absence or presence of 0.5 mg/mlHIP peptide. 0.5 μg of digested HS was injected into capillary HPLCcoupled with mass spectrometry (FIG. 8). UV peak B eluted at the sametime as a ΔUA-GlcNS6S standard, whereas UV peak D eluted at the sametime as a ΔUA2S-GlcNS standard (FIG. 8, panel A). Three major ions withm/z 247.5, 496.0, and 625.2 were observed in both UV peaks (FIG. 3,panel B and D), where 496.0 is z1 (−1) charged, 247.5 is z2 (−2)charged, and 625.2 is one dibutylamine adducted, z1 (−1) chargedΔUA-GlcNS6S or ΔUA2S-GlcNS disaccharides. However, when m/z regions494.0 to 501.0 from both peal B and peak D were expended (panel C andpanel E), a non-natural abundant, z1 charged molecular ion with m/z498.0 was observed in UV peal B, but not in UV peak D. 498.0 vs. 496.0of disaccharide ions should represent ΔUA-GlcNS3[³⁴5]S and ΔUA-GlcNS6S,respectively. The mass for ΔUA-GlcNS3[³⁴]S is barely detectable in theabsence of HIP peptide, which is consistent with the literature that3-O-sulfate containing sugars are usually degraded into tetrasaccharidesnot disaccharides by a mixture of heparitinase digestion (20,33). HIPpeptide was included in heparitinase digestion when 3-O-containing HSwere degraded into disaccharides.

Materials and Methods for Practicing the Inventions Exemplified Above

Cell Culture. Wild-type Chinese hamster ovary cells (CHO-K1) wereobtained from the American Type Culture Collection (CCL-61; ATCC,Rockville, Md.). CHO cells were maintained in Ham's F-12 mediumsupplemented with 10% fetal bovine serum (HyClone), penicillin G (100units/ml), and streptomycin sulfate (100 μg/ml) at 37 C under anatmosphere of 5% CO₂ in air and 100% relative humidity. The cells werepassaged every 34 days with 0.125% (w/v) trypsin and 1 mM EDTA, andafter 10-15 cycles, fresh cells were revived from stocks stored underliquid nitrogen. Low-sulfate medium was composed of Ham's F-12 mediumsupplemented with penicillin G (100 units/ml) and 10% fetal bovine serumthat had been dialyzed 200-fold against phosphate-buffered saline (30).Low-glucose Ham's F-12 medium contained 1 mM glucose supplemented withpenicillin G (100 units/ml), streptomycin sulfate (100 μg/ml), and fetalbovine serum that had been dialyzed 200-fold against phosphate-bufferedsaline (30). All tissue culture media and reagents were purchased fromLife Technologies (Gaithersburg, Md.) unless otherwise indicated.

3-OST-1 recombinant retroviral transduction. The retrovirus plasmidpMSCVpac was obtained from Dr. Robert Hawley, University of Toronto(31). pCMV3-OST-1 was digested with BglII and XhoI to release thewild-type murine 3-OST-1 cDNA (15). The cDNA fragment (1,623 bp) wascloned into the BglII+XhoI sites in pMSCVpac. All plasmid DNA preparedfor transfection was made with the Invitrogen SNAP-MIDI kit according tothe manufacturer's directions. Infectious virions were produced bytransducing ecotropic PHOENIX packaging cells with recombinant provirusplasmids using the calcium phosphate transfection technique. Followingthe precipitation step, the cells were re-fed with 2 ml/well of fleshDMEM and incubated overnight. Viral supernatants were collected, eitherflash-frozen in liquid nitrogen, and stored at −80° C. or used directlyafter low-speed centrifugation.

Wild-type CHO cells containing ecotropic receptors were treated withtrypsin and then plated at 150,000 cells/well in a 6-well dish. One daylater, target cells (<70% confluent) were incubated overnight with viralsupernatants containing 5 μg/ml Polybrene surfactant. After 12 hours,the virus containing media was replaced with fresh growth media.Wild-type CHO cells were exposed to recombinant retrovirus three timesand selected and maintained in 7.5 μg/ml puromycin (Sigma).

Antithrombin and FGF-2 labeling. The standard reaction mixture forpreparing fluorescent AT contained 20 mM NaH₂PO₄ (pH 7.0), 0.3 mM CaCl₂,25 μg of PBS dialyzed AT (GlycoMed), 4 mU neuraminidase (WorthingtonBiochemical Corp.), 4 mU galactose oxidase (Worthington BiochemicalCorp.), and 125 μg/ml fluorescein hydrazide (Molecular Probe, C-356) ina final volume of 280 μl. The mixtures were incubated at 37° C. for 1 h.PBS (1 ml) and a 50% slurry of heparin-Sepharose in PBS (100 μl) wasadded and mixed end-over-end for 20 min. After centrifugation, theheparin-Sepharose beads were washed 4 times with PBS (1 ml). Labeled ATwas eluted with four 0.25 ml aliquots of 10× concentrated PBS anddesalted by centrifugation for 35 minutes at 14,000 rpm through twoMicrocon-10 columns (Millipore). The concentrated AT was diluted with0.5 ml 10% FBS in PBS containing 2 mM EDTA and used directly for celllabeling studies.

Fluorescent FGF-2 was prepared by mixing 50 μl of 1 M sodium bicarbonateto 0.5 ml of PBS containing 2 mg/ml BSA and 3 μg FGF-2. The mixture wasthen transferred to a vial of reactive dye (Alexa 594, Molecular Probes)and stirred at room temperature for 1 hour. The isolation of the labeledFGF-2 was identical to that described above for labeled AT.

Cell sorting. Nearly confluent monolayers of 3-OST-1 transduced CHO K1cells were detached by adding 10 ml of 2 mM EDTA in PBS containing 10%FBS and centrifuged. The cell pellets were placed on ice and 50 μl eachof fluorescein-AT and Alexa 594-FGF-2 were added. After 30 minutes, thecells were washed once and resuspended in 1 ml of 10% FBS in PBScontaining 2 mM EDTA. Flow cytometry and cell sorting was performed onFACScan and FACStar instruments (Becton Dickinson) using dual colordetection filters. AT and FGF-2 binding positive cells were sorted andsubsequently single-cell cloned into a 96 well plate. The single cellclones were expanded and frozen for further analysis.

Twelve 3-OST-1 transduced CHO K1 clones were obtained as describedabove. The number of copies of 3-OST-1 in the individual clones wasdetermined by Southern analysis as follows. Genomic DNA (10 μg) wasdigested with 40 U of EcoRI overnight at 37° C., electrophoresed on a0.7% (w/v) agarose gel, transferred to GeneScreen Plus (NEN) and probedwith 3-OST-1 cDNA labeled with the Megaprime labeling kit (Amersham).Blots were hybridized in ExpressHyb Solution (Clontech) containing3-OST-1 probe (2×10⁶ cpm/ml), followed by autoradiography. The cellclone with 3 copies of 3-OST-1 was expanded and frozen for furtherstudies.

Mutant screening. Wild-type CHO with 3 copies of 3-OST-1 weremutagenized with ethylmethane sulfonate as described in the literature(31) and frozen under liquid nitrogen. A portion of cells was thawed,propagated for 3 days, and labeled with both Alexa 594-FGF-2 andfluorescein-AT. The labeled cells were sorted and FGF-2 positive and ATnegative cells were collected. Approximately 1×10⁴ sorted cells werecollected into 1 ml of complete F-12 Ham's media, then plated in T-75flasks. Sorted cell populations were maintained in complete F-12 Ham'smedium for one week, then the cells were labeled and sorted again asdescribed above. After 5 rounds of sorting, FGF-2 positive and ATnegative cells were single-cell-sorted into a 96 well plate. The singlecell clones were expanded and frozen for further analysis. The sortingprofiles of CHO K1 with 3 copies of 3-OST-1, precursor mutant, and the6-OST-1 correctant of the mutant were shown by dual-color fluorescenceflow cytometric analysis in FIG. 5.

HS Preparation and analysis. Cell monolayers were labeled overnight with100 μCi/ml of carrier free sodium [³⁵S]sulfate (ICN) in sulfatedeficient DMEM, supplemented with penicillin G (100 Units/ml), and 10%(v/v) dialyzed FBS. The proteoglycan fraction was isolated byDEAE-Sepharose chromatography and beta-eliminated in 0.5 M NaBH₄ in 0.4M NaOH at 4° C. overnight. The samples were neutralized with 5 M aceticacid until bubble formation ceased and the released chains were purifiedby another round of DEAE-Sepharose chromatography followed by ethanolprecipitation. The pellet from centrifugation was washed with 75%ethanol and resuspended in water. The GAGs were digested with 20 mU ofchondroitinase ABC (Seikagaku, Inc.) in buffer containing 50 mM Tris-HCland 50 mM sodium acetate (pH 8.0). Complete digestion of chondroitinsulfate by chondroitinase ABC was assured by monitoring the extent ofconversion of the carrier to disaccharides (100 μg=1.14 absorbance unitsat 232 nm). HS was purified from chondroitinase degraded products byphenol/chloroform (1:1, v/v) extraction and ethanol precipitation. Afterwashing the pellets with 0.5 ml of 75% ethanol, the HS was dissolved inwater for further analysis.

cDNA cloning and expression of CHO 6-OST-1. Sequences coding for CHO6-OST-1 were amplified from a CHO K1/cDNA quick-clone library(Clontech). The reaction mixture contained 2 units pfu polymerase(Stratagene), 1 ng of cDNA, and 100 pmol of the Primers. The senseprimer has an added Bgl II site (5′GCAGATCTGCAGGACCATGGTTGAGCG CGCCAGCAAGTTC-3′) (SEQ ID NO. 8) and the antisense primer has an added Xba Isite (5′-GCTCTAGACTACCACT TCTCAATGATGTGGCTC-3′) (SEQ ID NO. 9). The6-OST-1 primer sequences are derived from the human 6-OST-1 cDNAsequence (from residue 240 to 264) and to the complement of thissequence (from residue 1147 to 1172) as reported (32). After 30 thermalcycles (1 min of denaturation at 94° C., 2 min of annealing at 55° C., 3min of extension at 72° C.), the amplification products were analyzed in1% agarose gels and detected by ethidium bromide staining. Theamplification products were excised from the gel and cleaned by GelExtraction kit (Qiagen). The PCR product was treated with Bgl II and XbaI, ligated into Xba I and BamHI digested pInd/Hygro plasmid (Clontech)and transformed into E. coli DH5α competent cells. Four clones from eachof two separate PCR reactions were sequenced and found to be identical.pind/Hygro 6-OST-1 containing plasmid was transfected into the CHOmutant cells. AT and FGF-2 binding positive cells were sorted andsubsequently single-cell-cloned into a 96 well plate. The single cellclones were expanded and frozen for further analysis.

6-O-sulfation of HS in vitro. The standard reaction mixture contained 50mM MES (pH 7.0), 1% (w/v) Triton X-100, 5 mM MnCl₂, 5 mM MgCl₂, 2.5 mMCaCl₂, 0.075 mg/ml protamine chloride, 1.5 mg/ml BSA, eithermetabolically labeled [³⁵S]HS or non-radioactive HS chains, cold PAPS(0.5 mM) or [³⁵S]PAPS (25 μM, 2×10⁷ cpm), and 70 ng of purifiedbaculovirus-expressed human 6-OST-1 in a final volume of 50 μl. Themixtures were incubated either 20 minutes or overnight at 37° C., and200 μg of chondroitin sulfate C was added. HS chains were purified byphenol/chloroform extraction and anion exchange chromatography on0.25-ml columns of DEAE-Sephacel packed in 1 ml syringes (20). Afterethanol precipitation, the pellets were washed with 75% ethanol, driedbriefly under vacuum, and dissolved in water for further analysis.

Separation of HS^(act) and HS^(inact) by AT-affinity chromatography.AT-HS complexes were created by mixing 3-O-sulfated HS in 500 μl of HBbuffer (150 mM NaCl, 10 mM Tris-Cl (pH 7.4)) with 2.5 mM AT, 100 μg ofchondroitin sulfate, 0.002% Triton-X 100, and 1 mM each of CaCl₂, MgCl₂,and MnCl₂ (18). HB containing ˜50% slurry of Concanavalin A-Sepharose 4B(60 μl) was then added. AT complexes were bound to Concanavalin A by wayof the Asn-linked oligosaccharides. After one hour end-over-end rotationat 4° C., the beads were sedimented by centrifugation at 10,000×g. Thesupernatant was collected and the beads were washed three times with1.25 ml of HB containing 0.0004% Triton-X 100. The supernatant andwashing solutions contained HS^(inact). The HS^(act) was eluted withthree successive washes with 100 μl HB containing 1 M NaCl and 0.0004%Triton-X 100. After adding 100 μg of chondroitin sulfate as carrier toHS^(act), the sample was extracted with an equal volume ofphenol/chloroform, followed by chromatography on DEAE-Sepharose andethanol precipitation. The pellets were washed with 75% ethanol, driedbriefly under vacuum and dissolved in water.

Disaccharide analysis of HS. Heparitinase I (EC. 4.2.2.8), heparitinaseII (no EC number), and heparinase (EC. 4.2.2.7) were obtained fromSeikagaku heparitinase IV was obtained from Dr. Yoshida, SeikagakuCorporation, Tokyo. Heparitinase I recognizes the sequences:GlcNAc/NS±6S(3S?)-↓GlcUA-GlcNAc/NS±6S. The arrow indicates the cleavagesite. Heparitinase II has broad sequence recognition:GlcNAc/NS±6S(3S?)-↓GlcUA/IdoUA±2S-GlcNAc/NS±6S. Heparinase(heparitinaseIII) and heparitinase IV recognize the sequences:GlcNS±3S±6S-↓IdoUA2S/GlcUA2S-GlcNS±6S. The reaction products andreferences can be found in the following references (33,34). Thedigestion of HS^(act) was carried out in 100 μl of 40 mM ammoniumacetate (pH 7.0) containing 3.3 mM CaCl₂ with 1 mU of heparitinase I or1 mU of each heparitinase I, heparitinase II, heparitinase IV, andheparinase (heparitinase III). The digestion was incubated at 37° C.overnight unless otherwise indicated. For low pH nitrous aciddegradation, radiolabeled HS samples were mixed with 10 μg bovine kidneyHS (ICN) and digested (35).

Disaccharides were purified by Bio-Gel P2 chromatography and resolved byion pairing reverse-phase HPLC with appropriate disaccharide standards(36). Bio-Gel P2 or P6 columns (0.75×200 cm) were equilibrated with 100mM ammonium bicarbonate. Radiolabeled samples (200 μl) were mixed withDexdran blue (5 μg) and phenol red (5 μg) and loaded on the column. Thesamples were eluted at a flow rate of 4 ml/hour with collection of 0.5ml fractions. The desired fractions were dried under vacuum,individually or pooled to remove ammonium bicarbonate.

Capillary IPRP-HPLC coupled with mass spectrometry. Heparin molectilesexhibiting a high affinity for a synthetic peptide (CRPKAKAKAKAKDQTK) ISEQ ID NO. 7) mimicking a heparin-binding domain of heparin interactingprotein (HIP) also show an extremely high affinity for AT (37). It wasexpected that inclusion of this small peptide in the heparitinasedigestion solution would protect 3-O-[³⁵S]sulfate labeled HS fromdegrading into tetrasaccharide. Theoretically, HIP peptide-protected, ATbinding HS oligosaccharides would be recovered. However, in the presenceof the HIP peptide, all the 3-O-[³⁵S]sulfate labeled sugars weredegraded into disaccharides instead of oligosaccharides ortetrasaccharides as judged by their elution position on Bio-Gel P2 andtheir unique elution positions on IPRP-HPLC (the major 3-O-[³⁵S]sulfatecontaining disaccharides eluted right before ΔUA-GlcNS6S disaccharidestandard). Because there is no ΔUA-GlcNS3S standard reported, thestructure was verified. Stable isotope PAP³⁴S was made. The PAP³⁴S (99%isotope purity determined by ES-MS) was prepared by incubating ATP andstable isotope Na₂ ³⁴SO₄ (Isonics Corp.) with ATP sulfurylase (Sigma),adenosine 5′-phosphosulfate kinase (a generous gift from Dr. Irwin H.Segel), and inorganic pyrophosphatase (Sigma) (38). HS chains fromwild-type CHO cells were labeled with pure 3-OST-1 plus PAP³⁴S. Acapillary IPRP-HPLC (LC Packings) method for separating HS disaccharideswas developed. This method is similar to conventional IPRP-HPLC (29)except using 5 mM dibutylamine as an ion pairing reagent (Sigma), andthen coupled it to an ESI-TOF-MS (Mariner Workstation, PerSeptiveBiosystems, Inc.) to detect the mass of each disaccharide eluted. Six HSdisaccharide standards from Seikagaku were separated by capillary HPLCand detected by negative polarity ESI-MS. The accuracy of the ES-MS is±0.001 m/z unit after calibration with the molecular standard setssupplied by the manufacture (Bis TBA, Heptadecaflurononanoic acid,Perflurotetradecanoic acid). 3-O-³⁴S-labeled HS was digested with acombination of 1 mU of each heparitinase I, heparitinase II,heparitinase IV, and heparinase in the absence or presence of 0.5 mg/mlHIP peptide. 0.5 μg of digested HS was injected into capillary HPLCcoupled with mass spectrometry (FIG. 8). UV peal B eluted at the sametime as a ΔUA-GlcNS6S standard, whereas UV peak D eluted at the sametime as a ΔUA2S-GlcNS standard (FIG. 8, panel A). Three major ions withm/z 247.5, 496.0, and 625.2 were observed in both UV peaks (FIG. 3,panel B and D), where 496.0 is z1 (−1) charged, 247.5 is z2 (−2)charged, and 625.2 is one dibutylamine adducted, z1 (−1) chargedΔUA-GlcNS6S or ΔUA2S-GlcNS disaccharides. However, when m/z regions494.0 to 501.0 from both peak B and peak D were expended (panel C andpanel E), a non-natural abundant, z1 charged molecular ion with m/z498.0 was observed in UV peak B, but not in UV peak D. 498.0 vs. 496.0of disaccharide ions should represent ΔUA-GlcNS3[³⁴5]S and ΔUA-GlcNS6S,respectively. The mass for ΔUA-GlcNS3[³⁴S]S is barely detectable in theabsence of HIP peptide, which is consistent with the literature that3-O-sulfate containing sugars are usually degraded into tetrasaccharidesnot disaccharides by a mixture of heparitinase digestion (20,33). HIPpeptide was included in heparitinase digestion when 3-O-containing HSwere degraded into disaccharides.

Northern blot hybridization and RT-PCR. To generate specific Northernblot hybridization probes, PCR primers were designed that bracket uniquesequences within human 6-OST-1, 6-OST-2 and 6-OST-3. A 249 bp PCRproduct that corresponds to a region within the 3′-UTR of the 60ST-1gene starting at position 1772 and ending at 2021 was used as an isoformspecific probe. Similarly, a 299 bp PCR product that corresponds to aregion in the 3′-UTR of the 60ST-2 gene starting at position 1831 andending at 2130, and another product within the 3′-UTR of the 6-OST-3gene starting at 943 and ending at 1378 (444 bp) were used as a probe.PCR was performed with α[32P] dCTP (NEN Life Science Products) andisoform-specific radio-labeled probes were purified on G-25 Sephadexspin columns (Boehringer Mannheim). Hybridizations were carried out asto the manufacturer's instructions using 2×10⁶ cpm probe per ml ofExpressHyb solution (CLONTECH). After the hybridizations were complete,the blots were washed twice in 2×SSC containing 0.1% SDS and once with0.1×SSC containing 0.1% SDS, all at room temperature. Blots were thenwashed with 0.1×SSC containing 0.1% SDS at 50° C. For blots hybridizedwith the 6-OST probe, this last wash was repeated twice at 65° C. Themembranes were then subjected to autoradiography with BioMax imagingfilm (Kodak) with a BioMax MS intensifying screen (Kodak).

For RT-PCR, poly A purified or DNase I treated total RNA was used.Primer pairs were designed that bracket isoform specific regions withinthe human sequences for both 60ST-2, and 60ST-3. For 60ST-1, a 569 bpfragment corresponding to nt 54 (GCG TGC TTC ATG CTC ATC CT) (SEQ ID NO.10) to 622 (GTG CGC CCA TCA CAC ATG T) (SEQ ID NO. 11) within thehamster sequence was used. For 60ST-2, PCR targets included regionsstarting at nt 23 (CTG CTG CTG OCT TTG GTG AT) (SEQ ID NO. 12) and 346(GCA GAA GAA ATG CAC TTG CCA) (SEQ ID NO. 13) and ending at nt 1471 (GCCOCT ATC ACC TTG TCC CT) (SEQ ID NO. 14), 1491 (TCA TTG GTG CCA TTG CTGG) (SEQ ID NO. 15) and 1532 (TGA GTG CCA GTT AGC GCC A) (SEQ ID NO. 16).For 60ST-3, the targets included regions that start at nt 5 (CCG GTG CTCACT TTC CTC TTC) (SEQ ID NO. 17) and 353 (TTC ACC CTC AAG GAC CTG ACC)(SEQ ID NO. 18) and end at nt 988 (GCT CTG CAG CAG GAT GGT GT) (SEQ IDNO. 19) and 1217 (GCT GGA AGA GAT CCT TCG CAT AC) (SEQ ID NO. 20). TotalRNA was purified from wild-type and precursor mutant CHO-K1 cells usingthe RNeasy total RNA kit from Qiagen as to the manufacturer'sinstructions. RNA was quantitated by absorbance at 260 nm and 100 μg oftotal RNA was reacted with DNase I (Ambion) at 37° C. for 45 minutes,twice extracted with equal volumes of acid phenol/chloroform,precipitated in ethanol, and reconstituted in DEPC treated water.Further selection of poly-A plus RNA was carried out with the OligotexmRNA kit (Qiagen). RNA integrity was checked after electrophoresis on a1% agarose gel and all RT reactions were run with M-MLV reversetranscriptase (Ambion) according to manufacturer's instructions. PCR wasperformed with Super Taq polymerase (Ambion).

Baculovirus expression and Purification of 6-OST-1. Human 6-OST-1recombinant baculovirus was prepared using the pFastBas HT donor plasmidmodified by the insertion of honeybee mellitin signal peptide (36) andthe Bac-to-Bac Baculovirus expression system (Life Technologies, Inc.)according to the manufacturer's protocol, except that recombinant bacmidDNA was purified using an endotoxin-free plasmid purification kit(Qiagen, Inc.) and transfection of Sf9 cells was scaled up to employ 3μg of bacmid DNA and 6×10⁶ exponentially-growing cells in a 100-mm dish.At day three post-transfection, baculovirus was precipitated from themedium with 10% PEG, 0.5 M NaCl at 12,000×g, re-suspended in 14 ml ofmedium, and applied to a 100-mm dish seeded with 1.5×10⁷ Sf9 cells.Medium from the infected cells was harvested after 90 hours of growth at27° C., centrifuged at 400×g, made to 10 M in Tris, adjusted to pH 8.0,and centrifuged at 4000×g. Clarified medium was diluted with an equalvolume of cold 10 mM Tris-HCl, pH 8.0, and stirred for 30 minutes with0.6 ml (packed volume) of Toyopearl 650M chromatographic media(TosoHaas). The heparin-sepharose was packed into a column (0.4×4.75cm), washed with 5 ml of TCG 50 (10 mM Tris-HCl, pH 8.0, 2% glycerol,0.6% CHAPS, 50 mM NaCl), eluted with 1.2 ml of TCG 1000 (as above, but 1M in NaCl) containing 10 mM imidazole, and concentrated to 0.25 ml in aMicrocon YM-10 centrifugal filter (Millipore Corp.).

Histidine-tagged recombinant 6-OST-1 was affinity purified by mixing theproduct eluted from heparin-sepharose for 90 minutes at 4° C. with NiNTAmagnetic agarose beads (Qiagen, Inc.) and magnetically sedimented from60 μl of suspension. The beads were washed twice with 0.125 ml of TCG400 containing 20 mM imidazole and eluted twice with 0.03 ml of TCG 400containing 250 mM imidazole. The combined elution fractions containedapproximately 25% of the sulfotransferase activity present in thestarting medium.

Bacterial expression and Unification of 6-OST-1. Expression vectorpET15b was purchased from Novagen (Madison, Wis.). E. coli strains BL21and DH5α were obtained through ATCC (Manassas, Va.). An Ase Irestriction site was introduced at 211-216 bp and a BamHI restrictionsite was introduced at 1344-1349 bp of human 6-OST-1-1(32) by PCR. The6-OST-1 gene was then ligated into Nde I and BamHI digested pET15b andtransformed into competent E. coli strain DH5α. A BL21 colony containing6-OST-1 in pET15b with confirmed sequence was used to inoculate 2 L ofLB containing 100 μg/mL ampicillin. The cultures were shaken in flasksat 250 rpm at 37° C. When the optical density at 600 nm reached 1.2, 1mM IPTG was added to the cultures. The cultures were then agitated at250 rpm overnight at room temperature. The cells were pelleted at 5,000rpm for 15 minutes. The supernatant was discarded and the cell pelletwas resuspended in 40 mL of 20 mM Tris, 500 mM NaCl, 0.6% CHAPS, 1%glycerol, and 5 mM imidazole, pH 7.9 (“binding buffer”). The cells werehomogenized, and the homogenate was centrifuged at 13,000 rpm for twentyminutes. The supernatant was filtered through 0.2 μm filter paper andloaded onto a BioCAD HPLC system (PerSeptive Biosystems, Cambridge,Mass.) and purified using Ni²⁺ chelate chromatography. Briefly, thesupernatant was loaded onto the column and washed with binding bufferuntil unbound material was washed off the column. Then, low affinitymaterial was washed off the column using 20 mM Tris, 500 mM NaCl, 0.6%CHAPS, 1% glycerol, and 55 mM imidazole, pH 7.9 and 6-OST-1 was elutedfrom the column with 20 mM Tris, 500 mM NaCl, 0.6% CHAPS, 1% glycerol,and 500 mM imidazole, pH 7.9. The purity of the recombinant 6-OST-1 wasdetermined using a silver stained protein gel.

The invention disclosed herein may be embodied in other specific formswithout departing from the spirit or essential characteristics thereof.The foregoing embodiments are therefore to be considered in all respectsillustrative rather than limiting of the disclosed invention. The scopeof the invention is thus indicated by the appended claims rather than bythe foregoing description, and all changes which within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

The following references are incorporated by reference ill theirentirety.

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1. An in vitro method of transferring a sulfate on to the 6-O positionof a GlcNAc sugar residue in a polysaccharide preparation, the methodcomprising the steps of (a) providing a polysaccharide preparationhaving GlcNAc sugar residues, and (b) contacting the polysaccharidepreparation provided in (a) with glucosaminyl-6-O-sulfotransferase(6-OST) protein in the presence of a sulfate donor whereby the 6-OSTprotein adds a sulfate to the 6-O-position of a GlcNAc sugar residue;and wherein said polysaccharide preparation comprises heparin.
 2. Themethod of claim 1, whereby the polysaccharide preparation comprisesglucuronic acid (GlcUA) residues.
 3. The method of claim 1, whereby thepolysaccharide preparation includes GlcUA-GlcNAc 2 sugar residues. 4.The method of claim 1, whereby the polysaccharide preparation includesdisaccharides elected from the consisting of GlcUA/IdoUA-GlcNS,IdoUA2S-GlcNS, and GlcUA-GlcNS3S.
 5. The method of claim 1, whereby thepolysaccharide preparation includes GlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S.
 6. The method of claim 1, whereby thepolysaccharide preparation includes GlcNAc/NS-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S.
 7. The method of claim 1, whereby thepolysaccharide preparation includesGlcNAc/NS6S-GlcUA-GlcNS3S±-IdoUA2S-GlcNS6S.
 8. The method of claim 1,whereby the polysaccharide preparation includesGlcNAc/NS6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS.
 9. The method of claim 1,whereby the 6-OST protein is a recombinant protein.
 10. The method ofclaim 9, whereby the 6-OST protein is a human recombinant protein. 11.The method of claim 9, whereby the recombinant protein is produced in aexpression system selected from the group consisting of baculoviruscells, yeast cells, bacterial cells, and mammalian cells.
 12. The methodas in claim 1, whereby the sulfate donor is 3′-phospho-adenosine5′-phosphosulfate (PAPS).
 13. The method of claim 1, whereby the6-O-sulfation is performed in a reaction mixture comprising at least onechloride salt and wherein the pH is between 6.5 and 7.5.
 14. The methodof claim 1, whereby the polysaccharide preparation is contacted with6-OST protein in the presence of a sulfate donor for at least 20minutes.
 15. An in vitro method of enriching the portion ofanticoagulant active Heparin Sulfate (HS^(act)) present in apolysaccharide preparation comprising: (a) providing a d-O-sulfatedpolysaccharide preparation; and (b) contacting the preparation with6-OST protein in the presence of a sulfate donor under conditions, whichpermit the 6-OST protein to add a sulfate to the 6-O-position of aGlcNAc sugar residue, wherein step (b) occurs concurrent with orsubsequent to step (a).
 16. The method of claim 15, whereby the3-O-sulfated polysaccharide preparation is made in a CHO cell thatexpresses 3-OST-1 protein.
 17. The method of claim 16, whereby the3-O-sulfate polysaccharide preparation is prepared by contactinganticoagulant inactive Heparin Sulfate (HSin^(act)) with 3-OST-1protein.
 18. The method of claim 15, whereby the polysaccharidepreparation comprises heparan.
 19. The method of claim 15, whereby thepercentage of HS^(act) present in the polysaccharide preparationfollowing step (b) is greater than 70%.
 20. The method of claim 15,whereby the percentage of HS^(act) present in the polysaccharidepreparation following step (b) is greater than 50%.
 21. The method ofclaim 15, whereby the polysaccharide preparation compriseN-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) residues. 22.The method as in claim 15, whereby the polysaccharide preparationincludes sugar residues selected from the group consisting ofGlcUA/IdoUA-GlcNS, GlcUA-GlcNAc, IdoUA2S-GlcNS, and GlcUA-GlcNS3S. 23.The method of claim 15, whereby the sulfate donor comprises PAPS. 24.The method of claim 15, whereby the 6-O-sulfation is performed in areaction mixture comprising at least one chloride salt at a pH of about6.5-7.5.
 25. The method as in any one of claim 24, whereby the 6-OSTprotein comprises a polypeptide selected from the group consisting of(a) human 6-OST-1 (SEQ ID NO. 3); (b) human 6-OST-2A (SEQ ID NO. 4); (c)human 6-OST-2B (SEQ ID NO. 5); (d) human 6-OST-3 (SEQ ID NO. 6).